Neutrophils as major drivers of increased atherosclerosis in a murine model of chronic colitis

Abstract Background Recent findings showed a higher risk of premature atherosclerosis and cardiovascular events in patients with inflammatory bowel disease (IBD) especially during acute flare of the chronic disease. The underlying mechanisms remain to be defined since traditional risk factors such as hypercholesterinemia are not present in these patients. Purpose The present study aimed to unravel the underlying pathomechanisms of enhanced atherogenesis and -progression in a murine model of colitis. Methods Chronic colitis was induced in male Apolipoprotein E-deficient mice (Apoe−/−) using dextran sodium sulphate (DSS) in drinking water for 2,3 or 5 cycles, while the control group received regular drinking water. Each cycle consisted of 6 days DSS application and two weeks of recovery. Aortic atherosclerotic plaque burden was determined by en face Oil Red O staining and immune cell subsets were analysed by flow cytometry in the circulation, the bone marrow, and the aorta. Neutrophil depletion was performed via i.p injection of a Ly6G-depleting antibody or respective isotype control. Bone marrow-derived neutrophils were further analysed by transcriptome analysis. Results Similar to IBD patients, mice with chronic colitis exhibited an increased aortic plaque burden after 15 weeks of treatment despite the absence of classical risk factors. Over time, both circulating and aortic neutrophils showed an oscillatory detection pattern with the first significant increase after the 2nd DSS administration whereby the second increase after the 3rd DSS cycle was even stronger. Also, pro-inflammatory cytokines were elevated in the plasma and specifically G-CSF showed the same oscillatory pattern with increased plasma level already after the 2nd DSS administration and an even stronger increase after the 3rd thereby pointing towards alterations in bone marrow hematopoiesis. In line, flow cytometric analyses confirmed a greater rise of hematopoietic stem and even myeloid progenitor cells compared with the 2nd DSS application in IBD mice after 3rd treatment. scRNA-Seq analysis of progenitor cells revealed changes in cell differentiation towards neutrophils and upregulation of proinflammatory genes in isolated neutrophils of DSS-treated mice. These neutrophils showed also a more adhesive phenotype revealed by increased mRNA expression of Glg1 and Selplg. Accordingly, also Sele mRNA was increased in the aorta. The reduction of circulating neutrophils by an anti-Ly6G antibody during the acute phases of colitis reduced the aortic plaque burden compared to isotype treaded mice. Conclusion The current findings suggest detrimental effects of chronic colitis on atherogenesis and -progression in Apoe−/− mice via increased differentiation of myeloid cells into neutrophils and the promotion of a more adhesive and proinflammatory phenotype. These modified neutrophils may act as initiators of atherogenesis by promoting the invasion of immune cells into the aortic wall. Funding Acknowledgement Type of funding sources: None.